The blood-brain barrier (BBB) is composed of a specialized class of endothelium that forms a cellular barrier between the bloodstream and the interstices of the adult brain. By restricting non-specific flux of blood-borne constituents, the BBB plays an important role in maintaining parenchymal homeostasis, and strictly regulates transport of ions, small molecules, proteins, and cells into and out of the brain. The BBB accomplishes these tasks because its unique endothelium is endowed by epithelial-like tight junctions joining adjacent endothelial cells, lacks fenestrae, and possesses a rich array of molecular transport systems. Although the endothelium is the principle determinant of barrier function, perivascular non-endothelial cells in the local microenvironment have been shown to make significant contributions. Astrocytes (Stewart and Wiley 1981; Risau et al. 1986b; Janzer and Raff 1987), neurons (Tontsch and Bauer 1991) and pericytes (Balabanov and Dore-Duffy 1998; Ramsauer et al. 2002) have all been demonstrated to provide cues that result in the unique BBB endothelial phenotype.
Although the inductive properties of the aforementioned brain cell types have been confirmed through a multitude of in vivo and in vitro studies, the cell type(s) responsible for early embryonic BBB induction have not been distinguished. The developmental timecourse of embryonic BBB formation differs between species, but it is generally well accepted that the onset of BBB development begins prenatally and is followed by a gradual maturation to full BBB function (Bauer and Bauer 2000; Engelhardt 2003). For example, in rodents, vascular fenestrae disappear, pinocytosis decreases, and vessels decrease in diameter between embryonic days E11 and E17 (Bauer et al. 1993; Stewart and Hayakawa 1994; Bolz et al. 1996). The onset of tight junction formation is detectable from day E15, and tight junctions continue to increase in complexity through postnatal day P1 (Butt et al. 1990; Schulze and Firth 1992; Bauer et al. 1995; Kniesel et al. 1996; Nico et al. 1999). During this time, the transendothelial electrical resistance (TEER) of pial vessels is intermediate between peripheral vessels and the adult BBB (Butt et al. 1990; Schulze and Firth 1992; Bauer et al. 1995; Kniesel et al. 1996; Nico et al. 1999). A combination of the aforementioned attributes serves to restrict passage of protein into the embryonic brain (Risau et al. 1986a; Bauer et al. 1995; Dziegielewska et al. 2000), while a gradual decrease in BBB permeability to small tracers such as inulin and sucrose begins during embryonic development and continues postnatally (Ferguson and Woodbury 1969). Finally, transporter expression at the BBB also evolves from embryonic to postnatal stages as a result of changing nutritional needs (Johanson 1989; Gerhart et al. 1997).
The early embryonic developmental timecourse for the BBB raises the question as to what inductive factors or cell types drive the endothelial differentiation process. As mentioned above, astrocytes have long been linked with induction of BBB properties by in vitro and in vivo experiments (Stewart and Wiley 1981; Risau et al. 1986b; Janzer and Raff 1987). However, angiogenic vessels invade the immature embryonic neural environment and begin establishing BBB characteristics well in advance of the onset of gliogenesis as defined by the presence of GFAP-positive astrocytes in rodent brain (E18, (LeVine and Goldman 1988)). In addition, the developing BBB vessels have little extracellular matrix with few astrocyte contacts even just days prior to birth (Caley and Maxwell 1970). In fact, for rodents, much of the astrocyte generation takes place postnatally during which time the astrocyte sheath that surrounds mature brain capillaries is developed (Johanson 1989). Therefore, it is unlikely that astrocytes function in the early BBB induction process, but instead other cell types may be responsible for the early onset of BBB properties.
NPC are a major cell type in the developing embryonic brain, and it was recently reported that the differentiation and morphology of NPC are influenced by endothelial cells (Shen et al. 2004). In co-culture with endothelial cells, NPC show reduced neurogenesis and elevated self-renewal (Shen et al. 2004). Neural progenitors have also been observed in contact with early postnatal blood vessels, and this was implicated as an early stage in astrocyte differentiation (Zerlin and Goldman 1997). In addition, when endothelial cells and neural stem cells are grown in direct contact, it was shown that the adult neural stem cells could even produce progeny that exhibited an endothelial phenotype (Wurmser et al. 2004). Finally, adult neural stem cells are often found localized in perivascular spaces of the brain such as the subventricular zone and hippocampus, and it is thought that the vasculature is an important part of the stem cell niche (Doetsch 2003b).
Given the cellular interplay in the endothelial cell to NPC direction, we examined herein whether NPC could also influence brain endothelial cell phenotype. In this specification, we demonstrate that NPC isolated from the E14 embryonic brain induced BBB properties in an in vitro model consisting of primary rat brain microvascular endothelial cells in co-culture with NPC. We disclose an improved BBB model and method for examining the permeability of the BBB to test compounds.